Abstract
Neutrophil extracellular traps (NETs) formation has been implicated in an increasing number of infectious and non-infectious pathologies. NETosis is a tightly regulated process; the end-stage and read-out is the formation of DNA strands extruded from the nuclei, and traditionally assessed by fluorescence microscopy. Since NETosis has emerged as a possible biomarker of the inflammatory process, there is a need for less time-consuming, consistent, and quantitative approaches to improve its application in clinical assessment of pro-inflammatory conditions. Imaging Flow Cytometry (IFC) combines features of conventional flow cytometry with qualitative power of fluorescence microscopy and has an added advantage of the capability of assessing the early processes leading up to extrusion of the DNA-scaffolded strands. We explored the optimal imaging-based tools that can be used to measure citrullination of H4 in early NETosis. IFC identified and quantified histone 4 citrullination (H4cit3) induced with several known NETosis stimuli (Ionophore, PMA, LPS, Hemin, and IL-8) following treatment periods ranging from 2 to 60 min. Its relationship with other alterations at nuclear and cellular level, such as nuclear decondensation and super-condensation, multi-lobulated nuclei vs. 1-lobe nuclei and cell membrane damage, were also quantified. We show that the early progress of the H4cit3 response in NETosis depends on the stimulus. Our method identifies fast (Ionophore and Hemin), intermediate and slow (PMA) inducers and shows that H4cit3 appears to have a limited contribution to both early LPS- and IL-8-induced NETosis. While this method is rapid and of a higher throughput compared to fluorescence microscopy, detection and quantification is limited to H4cit3-mediated nuclear events and is likely to be stimulus- and signaling pathway dependent.
Highlights
Neutrophils are key players in the innate immunity system
Changes in nuclear decondensation and histone H4 citrullination (H4cit3)-expressing neutrophils were assessed with imaging flow cytometry (IFC) within 60 min (60′) of treatment with the stimuli
A change in the nuclear morphology from multilobular and well-organized to swelled and fuzzy-looking has been regarded as an early marker of neutrophils undergoing NETosis [10]
Summary
Neutrophils are key players in the innate immunity system. Resting neutrophils patrol the bloodstream and are rapidly recruited to sites of infection and injury upon activation, contributing to host defense, and inflammation. Analysis of other parameters, such as nuclear super-condensation, presence of unsegmented nuclei, and cell membrane damage, can be further used to pinpoint toward additional NETs-related characteristics. These analytical parameters were established by using a pharmacological inducer (PMA), and LPS and IL-8 (inducers associated with infectious pathogens) and Hemin (inducer associated with hemolysis). Purified neutrophils were treated with Hemin, LPS, PMA, and IL-8 as described in Table 1 and apoptotic and necrotic cells were detected with a FITC Annexin V Apoptosis Detection Kit with 7-AAD (Biolegend, cat#: 640922). Single color controls for each marker were used as compensation controls and unstained
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